This invention relates generally to the field of drug delivery, more particularly to the delivery of therapeutic gene products by transformation of cells of the intestine.
Proteins are essential to all biological functions, from metabolism, to growth, to reproduction, to immunity. As such, they halve an important potential role as pharmaceutical agents for the treatment of a wide range of human diseases. Indeed, they have already been used to treat diseases such as cancer, hemophilia, anemia and diabetes successfully, and for a number of diseases are the only effective treatment.
Although protein drugs have enormous therapeutic potential, their more widespread use has been limited by several restrictive technical factors. First, proteins remain difficult and expensive to manufacture compared to other pharmaceuticals. Large-scale purification of proteins in bioactive form can be a limiting step in the commercialization of these drugs. Second, many proteins are metabolized or otherwise eliminated quickly in the patient. This results in the need for frequent re-administration. Finally, protein drugs generally must be given by injection. This increases the complexity and expense of the treatment, and the disagreeable nature of administration also limits potential clinical applications.
Delivery of therapeutic gene products (such as polypeptides for protein replacement therapy) by expression in cells transformed with a therapeutic gene product-encoding DNA has attracted wide attention as a method to treat various mammalian diseases and enhance production of specific proteins or other cellular products. This promising technology, often referred to as gene therapy, is generally accomplished by introducing exogenous genetic material into a mammalian patient""s cells. The introduced genetic material can be designed to replace an abnormal (defective) gene of the mammalian patient (xe2x80x9cgene replacement therapyxe2x80x9d), or can be designed for expression of the encoded protein or other therapeutic product without replacement of any defective gene (xe2x80x9cgene augmentationxe2x80x9d) Because many congenital and acquired medical disorders result from inadequate production of various gene products, gene therapy provides a means to treat these diseases through either transient or stable expression of exogenous nucleic acid encoding the therapeutic product.
Delivery of therapeutic gene products by expression in transformed cells can be accomplished by either direct transformation of target cells within the mammalian subject (in vivo gene therapy) or transformation of cells in vitro and subsequent implantation of the transformed cells into the mammalian subject (ex vivo gene therapy). A variety of methods have been developed to accomplish in vivo transformation including mechanical means (e.g., direct injection of nucleic acid into target cells or particle bombardment), recombinant viruses, liposomes, and receptor-mediated endocytosis (RME) (for reviews, see Chang et al. 1994 Gastroenterol. 106:1076-84; Morsy et al. 1993 JAMA 270:2338-45; and Ledley 1992 J. Pediatr. Gastroenterol. Nutr. 14:328-37).
As with all therapies, the therapy that is most easily administered, least expensive, and most likely to realize patient compliance is the therapy of choice. Intestinal gene therapy provides such a therapy in the realm of gene therapy techniques. The intestinal epithelium is a particularly attractive site for in vivo gene therapy, largely due to the ease of access via an oral or other lumenal route, thus allowing administration of the exogenous nucleic acid via non-invasive procedures. For example, the patient can simply take a pill composed of the exogenous nucleic acid or alternatively the exogenous nucleic acid formulation can be administered by some other non-invasive means (i.e., a means that does not require a major surgical procedure, such as endoscopic catheterization or rectal suppository incision).
However, past efforts to accomplish in vivo transformation of intestinal cells have met with severe obstacles. Because the field has been primarily concerned with long-term transformation and delivery of the therapeutic gene product of interest, most groups have shunned intestinal epithelial cells as targets for transformation due to the cells"" rapid turnover rate (2 to 4 days) (see, e.g., Sandberg et al. 1994 Hum. Gene Therap. 5:303-9). Efforts to achieve in vivo transformation may be further complicated by the mucus layer of the intestine, which is thought to block access of the gene therapy transforming formulation to the target cells (Sandberg et al., supra). The presence of high concentrations of DNAses in the intestinal tract is also thought to be a formidable barrier to the effective introduction of DNA into intestinal tract cells.
Many of the vectors and delivery systems developed for in vivo cellular transformation either have their own inherent drawbacks or are not entirely suitable for in vivo intestinal cell transformation. For example, recombinant viruses, particularly retroviruses, may be slow in gaining FDA approval due to concerns generally associated with the administration of live viruses to humans. In addition, it has become clear that viral vectors present problems with the possibility of multiple administrations of the gene construct due to immune responses, and may greatly limit their utility. Mechanical means, such as tie gene gun, are designed for use in transformation of skeletal muscle cells and are not particularly useful in intestinal cell transformation due to problems of access and to the delicate nature of organ.
Current methods for drug delivery by transformation that are designed to accomplish systemic therapeutic goals (e.g., to accomplish administration of protein-based drugs) include both ex vivo and in vivo techniques. However ex vivo techniques require complex procedures to accomplish transformation, put the subject at risk of rejection of the transplant, require at least minor invasive procedures, and limit implantation to modest numbers of cells. In vivo methods (e.g., direct administration through blood or to muscle) also frequently require invasive procedures and meet with difficulties in delivery of the transforming material to the target cell. Moreover, delivery of the transforming material via the bloodstream of the individual results in exposure of the DNA and any carrier associated with it to the immune system, which can result in adverse reactions (e.g., inflammatory reactions to the DNA administered and/or to components of the formulation containing the DNA).
Today, as the biomedical research enterprise discovers new proteins at an increasing pace, and as known proteins become available as therapeutic agents, there is a vital need to develop new delivery systems and methods to expand the application of these molecules as drugs by improving the feasibility and convenience of their use. The present invention addresses these problems.
Intestinal epithelial cells are genetically altered by exposure to a formulation comprising nucleic acids (including DNA, RNA, DNA-RNA hybrids, oligonucleotides, and synthetic nucleic acids), where such exposure results in the nucleic acid being operatively incorporated into the intestinal epithelial cell, thereby facilitating expression of a gene encoding a therapeutically effective protein. The nucleic acid formulation can be any formulation suitable for delivery to the gastrointestinal tract for introduction of the nucleic acid in the formulation into an intestinal epithelial cell including viral vectors, naked nucleic acid, and/or liposome formulation, preferably naked nucleic acid. More particularly, cells of the intestine are genetically altered to operatively incorporate a functional exogenous DNA sequence which, when expressed, produces a protein that is secreted directly from the cell into the bloodstream and/or into the gastrointestinal system in an amount sufficient to obtain therapeutic levels of the protein, thereby treating the patient in need of the protein. The method of the invention can thus be used to genetically alter cells to accomplish systemic therapy and/or repair of defects in the transfected cell itself.
A primary object is to provide a method of therapeutic gene product (e.g., protein) delivery wherein cells of the intestinal epithelium (e.g., cells of the small intestine or the large intestine) of a mammal are genetically modified by the incorporation of fully functional genes (exogenous DNA) which express a biologically active and therapeutically useful protein, which protein is secreted from the modified cells into the circulatory system, the gastrointestinal tract, and/or the local environment of gastrointestinal tissue.
Another object is to produce genetically transformed intestinal epithelial cells which have incorporated into their genome exogenous genetic material in the form of a fully functional gene which expresses a biologically active and therapeutically useful protein that functions within the cell. Alternatively, the transfected nucleic acid can provide structural, enzymatic or other direct intracellular effects independent of coding for a gene product. Examples include anti-sense nucleic acids that effect the regulation and expression of endogenous genes, and ribozymes which are nucleic acid sequences having intrinsic enzymatic activity.
The present invention is advantageous in that it exploits the enormous ability of cells lining the GI tract to produce and secrete proteins. The intestines are the second largest organ of the body, and the largest immune organ. The cells of the intestinal wall provide an interface with tremendous surface area (approx. 300 m2) that has as its normal function the preferential absorption of substances from the GI tract and transfer into the bloodstream this is not the case for lung or muscle tissues. Making use of even a small fraction of this capacity can provide many important therapeutic proteins, such as hormones, cytokines, and clotting protein, to the bloodstream. Moreover, short-acting proteins can be used with greater therapeutic effect as the present invention ensures their continuous synthesis and secretion at the needed rates.
An important advantage of the present invention is that it allows administration of protein drugs by mouth. Rather than attempting to deliver the protein itself, the delivery system of the invention does so indirectly by administering the gene encoding the therapeutic proteins. Despite the conventional wisdom that any DNA in the gastrointestinal tract would be destroyed rapidly by the digestive process (either by stomach acid or intestinal DNAse), oral delivery of DNA encoding a desired therapeutic protein is successful in the present invention. The DNA is taken up by intestinal cells, which synthesize the encoded protein and secrete it into the bloodstream or the gastrointestinal tract to achieve therapeutic results. The flexibility of this technology allows for the delivery of a wide variety of protein pharmaceuticals, systemically, into the gastrointestinal tract, as well as locally, making it well suited for a broad spectrum of therapeutic applications.
Another advantage of the present invention is that the short term expression of the therapeutic gene in the individual allows for regulation of administration of the therapeutic gene product to the patient. Because intestinal cells turn over rapidly, expression can be easily modified or altered by varying the dose and/or formulation of the oral preparation. Short-term expression is thus a consequence of the rapid turnover of transformed cells that are normally lost (or xe2x80x9cturned overtxe2x80x9d) within about two or three days. This aspect of the invention is both advantageous for dose control and reduction of risk of long-term complications from DNA integration (mutagenesis).
Another advantage of the invention is that the method completely avoids invasive procedures, and allows the vector to be administered in the simplest possible fashion-by the oral administration of a pill or other material. The lumen of the gastrointestinal tract is actually xe2x80x9coutsidexe2x80x9d the body, and is separated from it by a single continuous layer of cells. As such, anything that passes into the gastrointestinal tract through the mouth remains in the exterior space, and cannot enter the body proper and its bloodstream, unless it first crosses the cells that line the gastrointestinal tract. However, once the gene is expressed within the intestinal cells and the protein product released into the bloodstream via natural secretory pathways, the therapeutic protein acts in the same manner as current, injectable forms of the drug.
The present invention is also advantageous over gene-based therapies that administer the gene vector into the bloodstream to other tissues and organs in that it involves administration of the DNA of interest directly to the target cells of the subject without first being distributed broadly via the bloodstream. Thus delivery of the DNA using the invention is more efficient and avoids the need for additional mechanisms to target the DNA of interest to a particular tissue.
Still another advantage is that the present invention minimizes the exposure of the transforming DNA to the bloodstream, the major source of adverse reactions to treatment. Reaction by the immune system to the delivery vector (particularly viral vectors) is a major obstacle for conventional gene-based therapies. Delivery of vectors by other routes (e.g., intravenous, intramuscular injection, or pulmonary administration) exposes them to blood and extracelluar fluid. This exposure commonly results in inflammation and an immune response. These adverse reactions often worsen with reapplication, to the point where treatment cannot continue and becomes completely ineffective. The present invention presents the vector directly to the intestines without having to pass first through the blood or tissue of the subject. This shields the DNA delivery process as much as possible from the systemic circulation where immunological and inflammatory responses are initiated, and in this way minimizes their interference with therapy.
Another advantage of the invention is that naked DNA can be used as the vector, rather than viral vectors. Although viral vectors have been popular for gene therapy due to ease of administration and the incorporation of DNA into the genome, viral vectors have been found to produce substantial antigenic reactions that prevent their multiple administration. Use of naked DNA avoids this problem.
Yet another advantage of the invention is that potential deleterious side-effects of long term gene administration can be avoided, because the epithelial cells are sloughed into the intestinal lumen and lost from the body within a few days.
Another advantage of the invention is that the therapeutic gene product is delivered to the bloodstream of the subject in a manner and dosage that is more akin to normal production of the gene product (e.g., relative to bolus intravenous injection of a therapeutic polypeptide).
Still yet another advantage is that the drug delivery system of the invention uses the patient""s own tissue to produce the protein drug of interest and secrete it into the bloodstream.
Another advantage of the invention is that administration of DNA formulations directly to the lumen of the GI tract (rather than to the bloodstream) allows for the use of a greater variety of transfection adjuvants. As a result of its physiological role, the gastrointestinal tract is designed to be more robust, and is more able to tolerate a wider range of environmental conditions and less susceptible to toxic reactions. Thus the present invention can be used in conjunction with chemical methods to facilitate DNA uptake by cells that are suitable alternative to viral-based vectors, but may be otherwise unsuitable to administration via intravenous, intramuscular, or pulmonary routes. For example, since the GI tract is less susceptible to toxicity and does not require targeting from the circulation, the present invention allows expanded use of liposomes, adjuvants that are comprised of cationic lipids and enhance DNA uptake, as well as a variety to other adjuvants.
The invention is also advantageous in that it avoids many of the technical barriers associated with delivery of protein-based drugs. First, the invention avoids the cost and difficulty of manufacture of proteins by using the body""s own tissues to synthesize the desired proteins. Second, the invention avoids the problems of rapid metabolism by providing for the continuous manufacture and secretion of the therapeutic protein by gene expression. Finally, oral administration avoids the need for injection to achieve therapeutic levels in the body.
These and other objects, advantages and features of the present invention will become apparent to those persons skilled in the art upon reading the details of the vectors, formulations, and methodology as more fully set forth below.